Dipeptides find uses in various fields. For example, the dipeptides are used as raw materials for pharmaceuticals and functional foods. Specifically, L-alanyl-L-glutamine is used as a component of serum-free media. L-alanyl-L-glutamine is also used as a component of solutions for infusion, because it is more stable and more soluble in water than L-glutamine.
The dipeptides are generally produced by chemical synthetic methods. However, such methods often require complicated steps. Examples of such method may include use of N-benzyloxycarbonylalanine (hereinafter referred to as “Z-alanine”) and protected L-glutamine (Bull. Chem. Soc. Jpn., 34, 739 (1961) and Bull. Chem. Soc. Jpn., 35, 1966 (1962)); use of Z-alanine and protected L-glutamic acid-γ-methyl ester (Bull. Chem. Soc. Jpn., 37, 200 (1964)); use of Z-alanine ester and non-protected glutamine (JP-1-96194A); and synthesis using a 2-substituted propionyl halide as a raw material via N-(2-substituted)-propionylglutamine derivative as an intermediate (JP-6-234715A).
However, all of these methods require introduction and elimination of protective groups or synthesis of intermediates, so that none of them are industrially advantageous and fully satisfactory.
As typical production methods for producing dipeptides with enzymes, there have been known a condensation reaction using an N-protected-C-nonprotected carboxyl component and an N-nonprotected-C-protected amine component (hereinafter, “reaction 1”) and a substitution reaction using an N-protected-C-protected carboxyl component and an N-nonprotected-C-protected amine component (hereinafter, “reaction 2”). An example of the reaction 1 is a method for producing a Z-aspartylphenylalanine methyl ester from Z-aspartic acid and phenylalanine methyl ester (JP-53-92729A). An example of the reaction 2 is a method for producing acetylphenylalanylleucinamide from acetylphenylalanine ethyl ester and leucinamide (Biochemical J., 163, 531 (1977)). There are very few reports as to use of an N-nonprotected-C-protected carboxyl component. An example of a substitution reaction using an N-nonprotected-C-protected carboxyl component and an N-nonprotected-C-protected amine component (hereinafter, “reaction 3”) includes, for example, a method for producing arginylleucinamide from arginine ethyl ester and leucinamide as described in WO90/01555. An example of a substitution reaction using an N-nonprotected-C-protected carboxyl component and an N-nonprotected-C-nonprotected amine component (hereinafter, “reaction 4”) includes, for example, a method for producing tyrosylalanine from tyrosine ethyl ester and alanine as described in EP-278787A. Production methods that can be the most inexpensive among these production methods are those using reactions that fall within the category of the reaction 4 in which the number of the protective groups in the components used is the smallest.
However, enzymes used in the conventional example of the reaction 4 (EP-278787A) include reagents of relatively expensive carboxypeptidase preparations derived from yeast that belongs to the genus Saccharomyces or fungi or plants. The produced dipeptides contain amino acids with relatively high degrees of hydrophobicity. EP-278787A discloses no method that uses an enzyme derived from bacteria or yeast other than that belongs to the genus Saccharomyces. Further, no method has been known that produces alanylglutamine or alanylasparagine that has a high hydrophilicity. Thus, development of a production method for such peptides on an industrial scale and at a reduced cost has been demanded.